The Journal of Neuroscience, March 25, 2015 • 35(12):5067–5086 • 5067

Cellular/Molecular Characterizing KIF16B in Neurons Reveals a Novel Intramolecular “Stalk Inhibition” Mechanism That Regulates Its Capacity to Potentiate the Selective Somatodendritic Localization of Early Endosomes

X Atena Farkhondeh,1 Shinsuke Niwa,1 Yosuke Takei,1 and Nobutaka Hirokawa1,2 1Department of Molecular and Cell Biology and Department of Molecular Structure and Dynamics, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan, and 2Center of Excellence in Genome Medicine Research, King Abdulaziz University, Jeddah 21589, Saudi Arabia

An organelle’s subcellular localization is closely related to its function. Early endosomes require localization to somatodendritic regions in neurons to enable neuronal morphogenesis, polarized sorting, and signal transduction. However, it is not known how the somatoden- dritic localization of early endosomes is achieved. Here, we show that the kinesin superfamily protein 16B (KIF16B) is essential for the correct localization of early endosomes in mouse hippocampal neurons. Loss of KIF16B induced the aggregation of early endosomes and perturbed the trafficking and functioning of receptors, including the AMPA and NGF receptors. This defect was rescued by KIF16B, emphasizing the critical functional role of the protein in early endosome and receptor transport. Interestingly, in neurons expressing a KIF16B deletion mutant lacking the second and third coiled-coils of the stalk domain, the early endosomes were mistransported to the axons. Additionally, the binding of the motor domain of KIF16B to microtubules was inhibited by the second and third coiled-coils (inhibitory domain) in an ATP-dependent manner. This suggests that the intramolecular binding we find between the inhibitory domain and motor domain of KIF16B may serve as a switch to control the binding of the motor to microtubules, thereby regulating KIF16B activity. We propose that this novel autoregulatory “stalk inhibition” mechanism underlies the ability of KIF16B to potentiate the selective somatodendritic localization of early endosomes. Key words: early endosome; KIF16B; neurons; receptor trafficking; stalk inhibition

Introduction diseases, including neurodegeneration, cancer, and developmen- Neurons are highly polarized cells with a long axon and short tal defects (Salinas et al., 2008; Sarli and Giannis, 2008; Wood et dendrites (Nicholls, 2001). Commensurate with specific roles of al., 2008; Gerdes et al., 2009). The intraneuronal localization of axons and dendrites, the localization of organelles and proteins some organelles, such as mitochondria, synaptic vesicle precur- within these structures is differentially regulated. Motor proteins sors, and RNA-protein complexes, are regulated by KIFs (Nan- determine the subcellular localization of many organelles in neu- gaku et al., 1994; Okada et al., 1995; Zhao et al., 2001; Kanai et al., rons (Hirokawa et al., 2010). The kinesin superfamily proteins 2004; Cai et al., 2005; Glater et al., 2006; Niwa et al., 2008). The (KIFs) are microtubule and ATP-dependent motor proteins, appropriate localization of kinesin motors and their cargoes is most members of which transport cargoes to the microtubules critical for cell function. plus-end (Vale, 2003; Verhey and Hammond, 2009; Hirokawa et The early endosome (EE) is a membrane-bound organelle al., 2010). Defects in motor transport are associated with many that serves as a hub for receptor sorting, trafficking, signal trans- duction, and protein degradation (Hoogenraad et al., 2010; Lasiecka et al., 2010). EEA1-positive EEs are mainly localized in Received Oct. 13, 2014; revised Jan. 20, 2015; accepted Feb. 19, 2015. Author contributions: A.F., S.N., Y.T., and N.H. designed research; A.F., S.N., and Y.T. performed research; A.F. the somatodendritic region of neurons (Wilson et al., 2000). contributed unpublished reagents/analytic tools; A.F., S.N., Y.T., and N.H. analyzed data; A.F., S.N., Y.T., and N.H. When axonal proteins are missorted to dendrites, they are re- wrote the paper. moved from the dendritic plasma membrane by endocytosis, This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan Grant- transported to somatodendritic EEs, and re-sorted to axons in-Aid for Specially Promoted Research to N.H. We thank the members of the N.H. laboratory for assistance and valuable discussions; Y. Noda, T. Ogawa, Y. Kanai, Y. Tanaka, H. Miki, R. Nitta, H. Yajima, K. Nakajima, K. Mitsumori, (Sampo et al., 2003). However, the molecular mechanism deter- and X. Fang for technical assistance; and H. Sato, H. Fukuda, and T. Akamatsu for help. mining the specific somatodendritic localization of EEs remains The authors declare no competing financial interests. unknown. Correspondence should be addressed to Dr. Nobutaka Hirokawa, Department of Molecular and Cell Biology, KIF16B, a member of the kinesin-3 family, was identified as Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: the plus-end motor protein that transports Rab5-positive EEs [email protected]. DOI:10.1523/JNEUROSCI.4240-14.2015 and Rab14-positive vesicles in non-neuronal cells (Hoepfner et Copyright © 2015 the authors 0270-6474/15/355067-20$15.00/0 al., 2005; Ueno et al., 2011). KIF16B has a conserved motor do- 5068 • J. Neurosci., March 25, 2015 • 35(12):5067–5086 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism J. Neurosci., March 25, 2015 • 35(12):5067–5086 • 5069

Here, using a variety of molecular and cell biology methods, we investigated the function of KIF16B in neurons. We show that the stalk domain of KIF16B brings a potential to determine the specific somatodendritic localization of EEs mediated by a novel intramolecular inhibitory mechanism.

Materials and Methods Cloning KIF16B and construct engineering. Human KIF16B (NM_ 001199865.1) was purchased from Thermo Scientific cDNA library, and PCRed using KOD-plus DNA polymerase (Toyobo). All PCR products were cloned into pEGFP-N1, pEGFP-C1, pIRES, or pPAmCherry1-N1 (Clontech). The 3xFLAG-N1 was derived from pEGFP-N1 by replacing the GFP with a 3xFLAG tag. The 2xFYVE was generated by PCR from an HGF-regulated tyrosine kinase substrate (Riken Fantom clone Movie 1. Dynamics of colocalized KIF16B and early endosomes in neurons. KIF16B-mCherry B230365P04) to serve as an endosomal marker and was cloned into (red) and GFP-2xFYVE (green) were cotransfected into hippocampal neurons and observed miRNA, pIRES, pEGFP-C1, and the pGEX-4T1 vectors. Deletion mu- using an LSM710 Duo confocal laser-scanning microscope (Carl Zeiss). The movie shows colo- tants were made by amplifying KIF16B nucleotides corresponding to calized KIF16B and EE bidirectional movements in the somatodendritic region. The cell body of residues 1–560, 1–643, 1–810, 1–936, 1–1085, 1–1096, 1–1116, 1117– the neuron is on the right, and the dendrites are on the top left. The time-lapse covers 5 min, 1266, 548–1266, 1–810, and 1117–1266 (⌬second and third coiled- ␮ with images acquired every 2 s. Scale bars, 10 m. coils), 809–1096, and a chimera was generated using the motor domain of KIF5C, 1–560, and the PX domain, 1117–1266, of KIF16B. All con- main, three coiled-coils in the stalk domain, and a PX domain. In structs were engineered by PCR and cloned into pEGFP-N1 and HeLa cells, KIF16B regulates the recycling and degradation of pEGFP-C1 vectors. L1197A/F1198A point mutations in the PX domain EGF-receptors by controlling the localization and function of EEs of full-length K1F16B (KIF16BFull) were generated using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) and cloned in (Hoepfner et al., 2005). A structural study has shown that the PX pEGFP-N1. domain binds directly to phosphatidylinositol-3-phosphate Antibodies. Anti-KIF16B antibody was generated by immunizing (PI3P) at the endosomal membrane (Blatner et al., 2007). Intro- rabbits with the synthetic peptide (CEFSPFFKKGVFDYSSHGTG) con- ducing a point mutation that abolishes the binding to PI3P jugated with keyhole limpet hemocyanin, using an Imject maleimide- changes the localization of KIF16B. In mouse embryonic fibro- activated immunogen conjugation kit (Pierce). After four injections, blasts, KIF16B transports FGF-receptor-carrying vesicles from serum was collected and affinity-purified with antigen peptides using the Golgi to the plasma membrane via EEs (Ueno et al., 2011). SulfoLink columns (Pierce). The efficiency of the generated anti-KIF16B KIF16B is also reportedly involved in tubule formation and fis- antibody was determined using Western blotting and immunostaining. sion by inducing early endosomal fusion (Skjeldal et al., 2012). The antibodies used for immunofluorescence, Western blotting, coim- Another study showed that KIF16B mediates the transcytosis of munoprecipitation, and microtubule binding assays were as follows: anti-␤3-tubulin (Sigma, 1:1000), anti-MAP2 (Abcam, 1:100), anti- transferrin receptors to apical recycling endosomes in epithelial GM130 (BD Biosciences, 1:1000), anti-synaptotagmin (Millipore Biosci- cells (Perez Bay et al., 2013). A recent paper suggested that ence Research Reagents, 1:500), anti-EEAI (sc-6415, Santa Cruz kinesin-3 motors undergo cargo-mediated dimerization around Biotechnology, 1:100), anti-FLAG M2 (Sigma, 1:1000), anti-␣-tubulin the neck coil segment, which results in processive motion (Sop- (Sigma, 1:1000), anti-IgG (Abcam, 1:2000), anti-␤-actin (Abcam, pina et al., 2014). 1:4000), anti-GFP (MBL-598, 1:2000), anti-GluR1 extracellular epitope (Santa Cruz Biotechnology, 1:200), anti-NGFR p75 extracellular epitope 4 (Millipore, 1:1000). All HRP-conjugated antibodies were purchased from GE Healthcare and used at a dilution of 1:10,000, and all Figure 1. KIF16B transports early endosomes in somatodendritic areas of neurons. A, Cul- AlexaFluor-conjugated antibodies were purchased from Invitrogen and tured hippocampal neurons immunostained with anti-KIF16B and anti-EEA1 antibodies. B, used at a dilution of 1:250. Representative Western blot showing the specificity of the generated anti-KIF16B antibody in Cell culture and immunocytochemistry. COS-7 cells were cultured in ␤ brain, hippocampal neurons (E16), and PC12 cells. -Actin was used as a loading control. C–E, DMEM containing 10% FBS at 37°C in a 5% CO2 atmosphere. Primary KIF16B-GFP was expressed in hippocampal neurons and stained with an anti-EEA1 (C) and an cultured neurons were obtained from 4 to 10 embryonic hippocampi at anti-synaptotagmin(D)antibody.Higher-magnificationimagesoftheareasindicatedinA,C,D 16 d postcoitum from mice of either sex and grown on LabTek four-well are found below their respective panels. A, C, Images highlighting the colocalization of KIF16B chambered coverslips (Nunc) containing MEM (Invitrogen) supple- with EEA1 along dendrites. Scale bars, 10 ␮m. E, Quantification of endogenous KIF16B and mented with B27 (Invitrogen) and GlutaMAX-I (Invitrogen). For im- KIF16B-GFPcolocalizationwithEEA1andsynaptotagmin.Colocalizationwasquantifiedusinga munofluorescence microscopy, neurons at 8–10 DIV were transfected pixel-by-pixel correlation between the green and red channels with ImageJ software. F–H, using a calcium phosphate protocol described previously (Jiang and Expression of KIF16B in somatodendritic regions of neurons. GFP control (F) and KIF16B-GFP- Chen, 2006) and were fixed 10–12 h after transfection with 4% PFA in positive neurons (G) stained with anti-␤3 tubulin and anti-MAP2, which are neuron and den- PBS, washed twice with PBS, incubated with 0.1% Triton X-100 (Sigma) drite markers, respectively. White arrows indicate axons. H, Representative fluorescence for 5 min, and blocked with 1% BSA in PBS. Cells were incubated with profilesalonganaxonanddendrite,shownindottedyellowandwhiteboxesinG,indicatethat primary antibodies overnight at 4°C. Incubation with Alexa-labeled sec- KIF16B-GFP is absent from axons and present in dendrites. Fluorescence profiles were scored ondary antibodies (Invitrogen) was performed for 30 min at room tem- usingCarlZeissLSMimagesoftware.Scalebars,10␮m.I–N,AnalysisofKIF16BandEEdynam- perature. For all knockdown experiments, KIF16B KD, control, and ics. I, J, Time-lapse images of bidirectional movement of KIF16B and the EEs along the dendrite rescued neurons were fixed 72 h after transfection. For cell surface eval- inarepresentativeneuron,showninMovie1,thatexpressedGFP-2xFYVEandKIF16B-mCherry. uation of receptors in KIF16B KD, control, and rescued neurons, the Arrowsindicatesomeofthecolabeledclusters.Scalebars,5␮m.K,L,Kymographsshowingthe neurons were not permeabilized after fixation because both anti-GluR1 bidirectional motility of colocalized clusters, indicated by arrows. M, Classification of motility and anti-NGFR antibodies have extracellular epitopes. However, to de- (pϭ0.96;␹ 2 test).N,Percentageoftheanterogradeandretrogrademergedmotileeventsin tect intracellular and extracellular signals of the receptors, neurons were somatodendritic regions according to the average velocity. Clusters that vibrated or did not permeabilized after fixation with 0.1% Triton X-100 in PBS buffer for 5 move were not counted. Values are mean Ϯ SD. Eighteen neurons from three independent min at room temperature, followed by blocking as above. Other cell experiments were analyzed. n.s., Not significant. See also Movie 1. transfections were performed using Lipofectamine 2000 (Invitrogen). 5070 • J. Neurosci., March 25, 2015 • 35(12):5067–5086 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism J. Neurosci., March 25, 2015 • 35(12):5067–5086 • 5071

RNAi assay using microRNA. The BLOCK-iT Pol II miR RNAi Expres- PNP and centrifuged at 50,000 rpm for 10 min at 27°C. The supernatant sion vector kit (Invitrogen) was used following the manufacturer’s pro- (S1) was stored at 4°C for use in further analysis. The pellet (P1) was tocol. Target sequences were designed using the Invitrogen website washed with PEM and centrifuged again at 50,000 rpm for 10 min at (https://rnaidesigner.invitrogen.com/rnaiexpress/). The knockdown ef- 27°C. S1 and P1 fractions were subjected to SDS-PAGE followed by ficiency of three sequences was tested using Western blot analysis of Western blot analysis. hippocampal neurons 72 h after transfection. From these three con- Live neuron microtubule binding assay. Mouse hippocampal neurons structs, one was selected. Generated 2xFYVE tagged with GFP was then were cultured as described above and plated on LabTek four-well cham- cloned into target miRNA, as an endosomal marker and a transfection bered coverslips (Nunc) containing MEM (Invitrogen) supplemented control. ␤-Tubulin served as a loading control in immunoblotting assays. with B27 (Invitrogen) and GlutaMAX-I (Invitrogen), and transfected Cell surface protein biotinylation analysis. For cell surface protein bioti- with plasmids encoding full-length pEGFP, or 1–810 ϩ 1117–1226- nylation analysis, hippocampal neurons cultured in 6-cm-diameter pEGFP. Four hours later, neurons were treated with 0.1 ␮g/ml dishes for 8 d were transfected with KIF16B-RNAi, the control construct, streptolysin-O in permeabilization buffer (PB: 25 mM HEPES/KOH, 115 ϩ or KIF16B-RNAi RNAi-resistant KIF16B and grown for 72 h. Accord- mM potassium acetate, 5 mM sodium acetate, 5 mM MgCl2, 0.5 mM EGTA, ing to the manufacturer’s protocol, the cell surface proteins were biotin- and 10 mg/ml BSA; pH 7.4) for 2 min. After washing three times with PB, ylated and extracted with a Cell Surface Protein Isolation kit (Pierce). neurons were incubated with PB containing 1 mM AMP-PNP at 37°C in

Extracted proteins were then analyzed using immunoblotting. The intra- a5%CO2 atmosphere and monitored after 10 min. To visualize micro- cellular protein ␤-tubulin was used as a control for transfection effi- tubules in live neurons, a special BiG (GaAsP) detector in a Carl Zeiss ciency, and anti-KIF16B was used as a knockdown efficiency control. LSM710 Duo laser scanning confocal microscope was used to achieve a Quantification of protein bands was conducted using the National Insti- high-resolution image. tutes of Health ImageJ gel analysis software. Coimmunoprecipitation. The 1–810-3xFLAG, EGFP-809–1096 (third Electrophysiology. Whole-cell patch-clamp recordings of mEPSCs were coiled-coil), and EGFP-1117–1226 (PX domain) were transfected into performed on 13–16 DIV primary hippocampal cultures. During record- COS-7 cells. After 14 h, cells were lysed in 10 mM HEPES at pH 7.4, 150 ings, neurons were continuously perfused with an extracellular solution mM NaCl, and 1% NP40 with a protease inhibitor mixture (Roche). containing (in mM) as follows: 140 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2,10 Coimmunoprecipitation was performed using anti-GFP antibodies. Af- HEPES, and 20 glucose, buffered to pH 7.3 with NaOH. The intracellular ter incubation for 1 h at 4°C, protein-G was added and incubation con- solution present in the pipette contained (in mM) the following: 115 tinued for 1 more hour. Samples were then centrifuged at 1000 rpm for 1 CsMeSO4, 20 CsCl, 10 HEPES, 0.6 EGTA, 2.5 MgCl2, 0.4 Na3GTP, 4 min at 4°C followed by three washes in lysis buffer. Input and pellet ␮ Na2ATP, and 10 sodium phosphocreatine. Picrotoxin (50 M) was used fractions were subjected to SDS-PAGE followed by Western blot analysis to block inhibitory synaptic transmission, and 500 nM TTX was used to using anti-GFP and anti-FLAG antibodies. block the generation of action potentials. Recordings were acquired with GST pull-down assay. Purified GST or GST-tagged 809–1096 proteins an Axopatch 1D amplifier (Molecular Devices). Glass electrodes were (10 ␮g) were preincubated with beads in lysis buffer (50 mM Tris, pH 7.4, pulled with a Sutter P-97 micropipette puller (Sutter Instruments) to a 150 mM NaCl, 10 mM MgCl , 10% glycerol, 1% Triton X-100, 1 mM DTT, ⍀ 2 tip resistance (R) 2–3 M . Data were filtered at 1 kHz, digitized at 2 kHz, various protease inhibitors, and 1 mM ATP or 1 mM AMP-PNP) for 1 h at and analyzed using the Mini Analysis Program (Synaptosoft). The 4°C. Extracts of COS-7 cells transfected with various GFP-tagged 1–810, threshold of the mEPSC amplitude was set at 5 pA. Recordings with Rs full-length, and 1117–1226 KIF16B fragments were first incubated with Ͼ20 M⍀ and those with Rs that changed by Ͼ20% were not included in beads in lysis buffer for 0.5 h. The cleared supernatants were then trans- the analysis. ferred to a new batch of beads, mixed with GST or GST-tagged 809–1096, Microtubule cosedimentation assay. The 1–810-pEGFP, 1–1096- and rotated for2hat4°C. After washing the beads three times with lysis pEGFP, full-length pEGFP, 1–810 ϩ 1117–1226-pEGFP, and myosin buffer, the proteins captured by the beads were eluted by boiling in the Vb-pEGFP (negative control) were transfected into COS-7 cells. Four- SDS-PAGE sample buffer, resolved by SDS-PAGE, and detected by im- teen hours later, the cells were lysed with PEM buffer (100 mM PIPES, 1 munoblotting with anti-GFP antibodies. mM EGTA, 1 mM MgSO4 containing 1% Triton X-100 and complete Cell imaging. Immunofluorescent samples were observed with an Ax- protease inhibitor, Roche) and then centrifuged at 15,000 rpm for 10 min iophot microscope equipped with an LSM510 or LSM710 Duo confocal at 4°C. The lysate was then supplemented with 1 mg/ml of a taxol- system (Carl Zeiss). For quantification, the pinhole was fully opened and stabilized microtubule solution, which was purified from porcine brain both the gain and offset were fixed. The brightest optical plane was used. as previously described (Kondo et al., 1994). The mixture was incubated Weaker fluorescent samples were observed first to determine conditions; at room temperature for1hinthepresence or absence of 1 mM AMP- stronger fluorescent samples were then observed. All images of hip- pocampal neurons were observed at 8–10 DIV and images for all knock- 4 down experiments at 11 DIV (72 h after transfection). The mean fluorescence intensity and signal density were quantified using ImageJ Figure 2. C terminus of KIF16B acts as the early endosome binding domain. A, Schematic software, and the images were processed using Photoshop 7.0 (Adobe). showing the constructs. Stars represent L1197A/F1198A point mutations. B–I, Mutants fused When calculating ratios between axonal tip and axon intensities, care was with GFP, expressed in hippocampal neurons, and then immunostained with anti-EEA1 and taken not to saturate the signal. The fluorescence profiles of cells were anti-␤3 tubulin (B, C, E) and in samples stained with anti-MAP2 (G–I). Full-length KIF16B scored using Carl Zeiss LSM image software. (KIF16BFull)(B)andC-terminalKIF16BPX(C)showapunctuateendosomaldistributionpattern Live imaging. To perform live imaging of GFP-2xFYVE and KIF16B- that colocalizes with EEs in the somatodendritic (SD) regions. D, Quantification of KIF16BFull mCherry constructs, 8–10 DIV hippocampal neurons were cotrans- and KIF16BPX colocalization with EEA1. Colocalization was quantified using a pixel-by-pixel fected with the indicated constructs. At 12 h after transfection, live correlation between the green and red channels with ImageJ software. E, KIF16B containing neurons were observed with a Carl Zeiss LSM710 equipped with a 37°C point mutations displays a diffuse distribution of the mutant throughout the neuron, with incubator. Movement of KIF16B and FYVE was monitored over time, clusteredEEsinSDregions.F,StatisticalanalysisofEEdensityin500pixelsofSDregionindicates and images were acquired every 2 s. For the determination of the veloc- a significant difference in EE density between the KIF16Bmut and both KIF16BFull and ities, images were acquired over 100 s. To conduct live imaging of KIF16BPX. G, I, KIF16B containing L1197A/F1198A point mutations in the PX domain and (H) KIF16B, RNAi and control vectors were transfected into 8 DIV hip- control GFP. Both display a diffuse localization. J, Quantification of relative fluorescence inten- pocampal neurons. After 72 h, live neurons were observed with the Carl sity of KIF16BPX, KIF16BFull, KIF16BPXmut, and control GFP-transfected neurons, normalized Zeiss LSM710 Duo. For rescue experiments, hippocampal neurons were to background signal. No significant difference was found between KIF16BPXmut and GFP cotransfected with RNAi vector and RNAi-resistant KIF16B for 72 h. The controlorKIF16BFullandKIF16BPXmut.Quantificationsofdensities(F)andfluorescencemean dynamics of FYVE were monitored over time, and images were acquired intensities (J) were performed with ImageJ software. Data are mean Ϯ SD. Eighteen neurons every 2 s. For KIF16B KD, images were acquired every 10 s to avoid fromthreeindependenttransfectionswereexamined.***pϽ0.001(F,Student’sttest;J,post bleaching the signal from the small endosomes. Velocities were deter- hoc test). n.s., Not significant. Scale bars, 10 ␮m. mined over 100 s. To visualize the motility of the EEs in the presence and 5072 • J. Neurosci., March 25, 2015 • 35(12):5067–5086 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism J. Neurosci., March 25, 2015 • 35(12):5067–5086 • 5073 absence of the inhibitory domain, 8–10 DIV hippocampal neurons were vious studies showing that KIF16B-GFP accurately reflects the cotransfected with inhibitory domain (ID) ϩ GFP-2xFYVE, whereas localization of endogenous KIF16B (Hoepfner et al., 2005; Blat- control neurons were transfected with only GFP-2xFYVE. After 12 h, the ner et al., 2007). Thus, we took advantage of this probe to observe neurons were observed with the Carl Zeiss LSM710 microscope. The the precise intracellular localization of KIF16B in cells using im- movement of FYVE was monitored over time, and images were acquired munofluorescence microscopy. every 2 s. For ID ϩ FYVE, acquisition was set to every 10 s to avoid Several types of neuronal KIFs have been reported: axon- bleaching. Velocities were calculated using images acquired over 100 s. For live imaging, the path of an individual cluster was traced and the specific KIFs, dendrite-specific KIFs, and ubiquitous KIFs (Setou distances were evaluated using the LSM710 software. Clusters that vi- et al., 2000; Nakata and Hirokawa, 2003; Jacobson et al., 2006; brated or did not move were not included in velocity quantification. Huang and Banker, 2012). To clarify the localization of KIF16B in ImageJ software and Photoshop 7.0 (Adobe) were used for analysis and neurons, KIF16B-GFP-expressing cells were stained with the graphical representations. dendritic marker MAP2 and a neuronal marker, anti-␤3 tubulin. GFP-expressing neurons were used as control (Fig. 1F). KIF16B- Results GFP was significantly localized in the somatodendritic regions of KIF16B targets early endosomes to somatodendritic regions hippocampal neurons, but not in the axons (Fig. 1G,H). EEA1- of neurons positive EEs were also specifically localized in the cell body and In this study, we characterized the functional role of KIF16B in dendrites, as shown in Figure 1A, C. These results indicate that neurons. Previous studies have shown that KIF16B binds to EEs both KIF16B and EEA1-positive EEs are specifically localized to in nonpolarized HeLa cells (Hoepfner et al., 2005; Blatner et al., the somatodendritic region of neurons. 2007). Thus, KIF16B is a good candidate motor protein for reg- Previous studies have used tandemly chained FYVE domains ulating the somatodendritic localization of EEs in neurons. First, fused with GFP (GFP-2xFYVE) as an EE marker because the we generated an anti-KIF16B antibody and demonstrated the FYVE domain is the C-terminal part of EEA1 protein that specif- specificity of this antibody in Western blots of brain, hippocam- ically binds to the PI3P of EEs and does not affect their localiza- pal neurons (E16), and PC12 cells, as well as in immunocyto- tion or function (Burd and Emr, 1998; Komada and Soriano, chemical assays (Fig. 1A,B). Cultured hippocampal neurons 1999; Ellson et al., 2001). Therefore, 2xFYVE generated from an were immunostained with the anti-KIF16B antibody and with an HGF-regulated tyrosine kinase substrate and fused with GFP anti-EEA1 antibody, a marker for EEs. As a result, endogenous served as an EE marker. The somatodendritic localization of KIF16B was observed to colocalize with the endogenous EEs in so- GFP-2xFYVE is almost identical to that of endogenous EEs, as matodendritic regions (Fig. 1A). When human KIF16B-GFP was revealed by the anti-EEA1 antibody, validating its use as an EE expressed in neurons, its localization was similar to that of endoge- marker in neurons (data not shown) (Wilson et al., 2000). We nous KIF16B (Fig. 1C,E). Because EEs are not synaptotagmin- conducted dual-color live imaging of hippocampal neurons positive (Hoopmann et al., 2010), we used synaptotagmin as a cotransfected with GFP-2xFYVE and KIF16B-mCherry to evalu- negative control and found that the KIF16B-GFP signal was dis- ate the movement and colocalization of KIF16B and EEs in neu- tinct from the synaptotagmin punctate labeling (Fig. 1D,E). Fur- rons. Time-lapse recordings, kymographs, and quantification thermore, the expression of KIF16B-GFP did not change the revealed bidirectional movement of KIF16B colocalized with EEs localization of EEA1-positive EEs, which continued to be local- in the cell bodies and dendrites (Fig. 1I–M; Movie 1). This bidi- ized in the somatodendritic regions. This is consistent with pre- rectional motion is consistent with the bidirectional polarity of microtubules (MTs) in somatodendritic regions. The distribu- 4 tion and dynamics were similar to those of endosomes in non- Figure 3. Knockdown of KIF16B perturbs the movement of early endosomes and causes neuronal cells, exhibiting a distinctive intracellular localization aggregation.A,B,Hippocampalneuronsweretransfectedwithcontrol-miRNA,KIF16B-miRNA, and both short- and long-range movements with a range of ve- or KIF16B-miRNA ϩ KIF16B-RNAi-resistant (rescue) and analyzed with Western blotting. A locities (Nielsen et al., 1999; Gasman et al., 2003; Hoepfner et al., representativeblotisshown,withanti-␤tubulinservingasaloadingcontrol.B,Quantification 2005). Analysis of vesicle velocities revealed biphasic slow and of the relative amount of protein using densitometry normalized to the control-miRNA corre- fast distributions, with no significant difference in anterograde sponding to A is shown as mean Ϯ SD from four independent experiments. Average knock- and retrograde directions (0.048 and 0.121 ␮m/s, respectively; down: 73%; average rescue: 81%. ***p Ͻ 0.001 (Welch’s t test). Protein densities were Fig. 1N). The range of KIF16B velocities may be impacted by the quantified using ImageJ analysis software. C, Schematic showing the GFP-2xFYVE insertion cargoes carried in the EEs and/or by associated proteins. A previ- used as a transfection and endosomal marker in KIF16B RNAi and control vectors. miRNA was ous study showed that the expression of RhoD and hDia2C re- inserted into the 3Ј-UTR of GFP-2xFYVE cDNA. D–G, Hippocampal neurons were transfected duces and regulates the motility of EEs through interactions with with control-RNAi, KIF16B-RNAi, or KIF16B-RNAi ϩ KIF16B-RNAi-resistant (rescue), and then fixed and stained with an anti-MAP2 antibody (red). D, Relative signal density of EEs in 500 the cytoskeleton in HeLa cells (Gasman et al., 2003). Collectively, pixelsofSDregionsnormalizedtothebackgrounddensitycorrespondingtoE.DataaremeanϮ our immunocytochemistry and live imaging results demonstrate SD.**pϽ0.01(Student’sttest).E,F,LocalizationoftheEEsasrevealedbyGFP-2xFYVE(green) that KIF16B comigrates with EEs in the somatodendritic regions in the cell bodies and along the dendrites. G, Average frequency of EEs in SD regions according of neurons. to the fluorescent distribution of each cluster, indicating accumulation of EEs in knockdown. H–P, Analysis of EE dynamics in control, KIF16B-RNAi, and rescued neurons revealed by GFP- The C terminus of KIF16B is the early endosomal binding 2xFYVE(green).H–M,Time-lapseimagesandkymographs.H,I,Bidirectionalmovementofthe domain EEs along a dendrite in a control neuron. Arrows indicate a representative moving EE. J, K, To clarify the functional details of the KIF16B molecular motor KIF16B RNAi-treated neuron showing many nonmotile and aggregated EEs. Arrows indicate a protein in neurons, we sought to identify the EE binding domain. representativehaltedEE.L,M,RescuedneuronshowingrecoveredbidirectionalmotilityofEEs. We constructed KIF16B mutants and expressed them in neurons ArrowsindicatearepresentativerescuedandmovingEE.N,Classificationofmovement(control and knockdown: p Ͻ 0.001; control and rescue: p Ͻ 0.82; ␹ 2 test). Average percentage of (Fig. 2A). The KIF16B PX-only mutant (KIF16BPX-GFP; Fig. anterograde (O) and retrograde (P) motile EEs in somatodendritic regions based on average 2A) displayed a somatodendritic endosomal distribution pattern velocity. Vibrating clusters were not counted. No significant difference between O and P (one- that colocalized with endogenous EEs, similar to full-length way ANOVA and post hoc test). Eighteen neurons from three independent experiments were KIF16B (KIF16BFull-GFP), indicating that the C terminus is suf- analyzed. Scale bars, 10 ␮m. ficient for EE association (Fig. 2B–D). In an alternatively spliced 5074 • J. Neurosci., March 25, 2015 • 35(12):5067–5086 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism J. Neurosci., March 25, 2015 • 35(12):5067–5086 • 5075 variant of KIF16B that was distinct from the one we used, L1248/ tions for control, RNAi, and RNAi-resistant transfected neurons. F1249 point mutations in the PX domain abrogate the binding of In control hippocampal neurons, EEs were uniformly distributed KIF16B to EEs in HeLa cells (Blatner et al., 2007). Hence, for in the somatodendritic regions (Fig. 3D–G). When KIF16B was further analysis, we introduced similar L1197A/F1198A point knocked down, EEs formed aggregates both in the cell body and mutations in the human PX domain to generate a mutant con- dendrites (Fig. 3D–G). In rescue experiments, cotransfection of struct, KIF16BPXmut-GFP. When this protein was expressed in RNAi-resistant KIF16B with the KIF16B RNAi vector largely pre- hippocampal neurons, the fluorescence signal of KIF16BPXmut- vented the aggregation of EEs (Fig. 3D–G). To confirm the im- GFP was diffuse, and EEs clustered in the cell body and dendrites, munostaining results in KIF16B RNAi-treated and control cells, in contrast to the even endosomal distribution pattern of full- we conducted time-lapse imaging. Time-lapse images and kymo- length KIF16B or the PX domain (Fig. 2E,F). Identical to the graphs revealed that the bidirectional motility of the EEs was localization of the GFP control, KIF16BPXmut-GFP was distrib- perturbed in KIF16B knockdown neurons compared with that in uted throughout the neuronal cytosol and did not display an controls (Fig. 3H–K). In the KIF16B knockdown neurons, the endosomal localization pattern (Fig. 2E,G–J). Collectively, these average number of motile EEs was markedly decreased and the data indicate that the PX domain of the C-terminal of KIF16B is labeled endosomes aggregated and were immobilized compared required for its association with EEs and that L1197 and F1198 with those in controls (Fig. 3N). The EE aggregation phenotype residues are critical for attachment. in KIF16B knockdown neurons was consistent with results in fungus (Ustilago maydis) showing that a kinesin-3 null mutation Knockdown of KIF16B perturbs the movement of results in immobile clusters of EEs in hyphal cells (Bielska et al., early endosomes 2014). Coexpression of RNAi-resistant KIF16B with the KIF16B Next, to determine the impact of KIF16B in neurons, knockdown RNAi vector restored the motility of EEs (Fig. 3L–N). The distri- was performed using RNAi, and the localization of EEs was ob- bution of the velocities was bimodal, with a slow and fast peak in served. KIF16B knockdown efficiency assessed with Western blot control, KIF16B knockdown, and rescued neurons (Fig. 3O,P). analysis showed that RNAi significantly inhibited the expression The effect of KIF16B knockdown on EE movement was not sig- of KIF16B. Moreover, this knockdown could be rescued by nificantly different between anterograde and retrograde direc- RNAi-resistant KIF16B (average knockdown: 73%; average res- tions. The percentage of motile EEs was as follows: control, 83%; cue: 81%; Fig. 3A,B). To serve as a transfection control and an EE knockdown, 22%; rescue, 68% (Fig. 3N–P). Together, these re- marker during knockdown, GFP-2xFYVE was cloned into the sults suggest that KIF16B is essential for EE transport in somato- target miRNA vector (Fig. 3C), with similar expression condi- dendritic regions of neurons.

4 KIF16B modulates receptor trafficking Because it is well known that EEs participate in receptor traffick- Figure 4. Loss of KIF16B results in altered GluR1 and AMPA receptor trafficking. A–I, Neu- ing, we studied the effects of KIF16B knockdown on cellular lo- rons expressing RNAi were permeabilized and costained with anti-GluR1 (red) and anti-MAP2 calization of receptors. We examined the distribution of (blue) antibodies. Localization of EEs revealed by the EE marker, GFP-2xFYVE (green). A, B, glutamate receptor 1 (GluR1), a subunit of the AMPA receptor, Representative neuron transfected with the control miRNA vector. Insets (magnified), Example in control and KIF16B knockdown neurons. The results of the ofnoncolocalizedGluR1withEEsinthecellbody.B,LowerdensityofcolocalizedGluR1withEEs immunocytochemical assays showed that the number of GluR1 along the dendrite. C, D, Representative neuron transfected with KIF16B miRNA (KD). Enlarge- clusters colocalized with EEs was increased, whereas the number ments represent an example of aggregated and colocalized GluR1s with EEs in the soma. D, of GluR1 clusters not colocalized with EEs was decreased, in Aggregation and an increased number of colocalized GluR1-EEs along the KD dendrite. E, F,A representative neuron cotransfected with the KIF16B miRNA ϩ KIF16B-RNAi-resistant (res- KIF16B knockdown neurons compared with those in control cue). Insets (magnified), Example of noncolocalized GluR1 with EEs in the cell body. F, Aggre- (Fig. 4A–D,G,H). These EE-GluR1 clusters aggregated in den- gation and a high density of colocalized GluR1s with EEs were inhibited along the dendrite by drites and the perinuclear region of the cell body (Fig. 4C,D,I). the KIF16B-RNAi-resistant construct. Somas are shown separately for EEs, GluR1s, and the These effects of KIF16B knockdown on GluR1 were largely merged image in the A, C, E (right side). B, D, F, Arrows indicate representative examples of avoided when the KIF16B miRNA vector was cotransfected with colocalized GluR1s and EEs. G, H, Quantifications of the total number of GluR1 clusters colocal- the KIF16B miRNA-resistant construct (Fig. 4E–I). Because ized and not colocalized with EEs: per 50 ␮m of dendrites (G) and per 30 ␮m 2 of soma (H). I, these results suggested an alteration in the trafficking of GluR1, Average frequency of GluR1-EE clusters in SD region according to the fluorescent distribution of we compared the surface expression of GluR1 in control and each cluster, showing accumulation of EEs in KD neurons. J, K, Neurons expressing the miRNA KIF16B knockdown neurons. Immunofluorescence staining of were stained with anti-GluR1 antibody without permeabilization to visualize the cell surface cell surface GluR1 revealed that the number of GluR1 receptors clusters. J, Representative images of dendrites expressing the control, RNAi vector, and rescue constructs show a reduced number of surface clusters in KD and a recovery in rescued neurons. on the cell surface was significantly reduced in KIF16B knock- Whitedottedoutlinesindicatethedendriticmembranes.K,Statisticalanalysisofthenumberof down neurons (Fig. 4J,K). This result was confirmed using sur- surface GluR1 clusters per 50 ␮m of dendrites. Images were analyzed with ImageJ software. face biotinylation, which showed that cell surface expression of Scale bars, 10 ␮m. L, M, Cell surface protein biotinylation analysis. L, Cell surface evaluation of GluR1 in KIF16B knockdown neurons was significantly reduced proteinsinKIF16B-RNAi,control,andrescuehippocampalneurons.M,Quantificationofprotein compared with that in controls (Fig. 4L,M). Cotransfection of relative density using densitometry normalized to the control. The absence of intracellular the KIF16B RNAi-resistant construct rescued the defect in cell ␤-tubulinverifiedthepurityofthecellsurfaceproteinfraction.Anti-KIF16Bwasusedasknock- surface GluR1 expression induced by KIF16B RNAi (Fig. 4J–M). down efficiency control. Protein densities were quantified using ImageJ software. N–Q, Elec- To investigate the physiological impact of the mislocalization trophysiologicalanalysisofAMPAreceptors.N,RepresentativetracesshowingmEPSCrecorded of the AMPA receptor, we performed whole-cell patch-clamp fromculturedhippocampalneuronsofcontrol-andKD-transfectedcells.O,AmplitudeofAMPA recordings of cultured hippocampal neurons. KIF16B knock- receptor-mediated mEPSCs (control: 14.7 Ϯ 2.6 pA; KD: 11.5 Ϯ 2.4 pA). P, Interevent interval of AMPA receptor-mediated mEPSCs (control: 0.32 Ϯ 0.06; KD: 0.39 Ϯ 0.09). Q, Cumulative down or the control vector was transfected into neurons and probability curves for amplitude of AMPA receptor-mediated mEPSCs. For all statistical analy- mEPSCs were recorded. We observed a reduction in mean am- ses, error bars indicate mean Ϯ SD. Fifteen neurons for each vector from three independent plitude and an increase in mean interevent interval of AMPA transfections were examined. *p Ͻ 0.05 (Student’s t test). **p Ͻ 0.01 (Student’s t test). receptor-mediated mEPSCs in neurons transfected with the ***p Ͻ 0.001 (Student’s t test). M, n ϭ 3 independent experiments; Welch’s t test. knockdown construct compared with those transfected with the 5076 • J. Neurosci., March 25, 2015 • 35(12):5067–5086 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism J. Neurosci., March 25, 2015 • 35(12):5067–5086 • 5077

control vector (amplitude: control, 14.7 Ϯ 2.6 pA; knockdown, structs and expressed them in neurons. Interestingly, when the 11.5 Ϯ 2.4 pA) (interevent interval: control, 0.32 Ϯ 0.06 s; knock- third coiled-coil of KIF16B was deleted (KIF16B936-GFP), the down, 0.39 Ϯ 0.09 s) (Fig. 4N–P). A cumulative probability curve protein accumulated in some neurites. MAP2 and tubulin-␤3 also indicated a leftward shift toward smaller amplitudes in staining revealed that KIF16B936-GFP specifically accumulated KIF16B knockdown neurons (Fig. 4Q). These data indicate a in the tips of axons (Fig. 6A,B,E). Thus, we set out to identify the decrease in AMPA receptor function in KIF16B knockdown region critical for the mislocalization of KIF16B. We made several neurons. mutants; and among them, only the KIF16B810-GFP mutant To confirm the role of KIF16B in receptor trafficking, we also lacking the second and third coiled-coils displayed a localization examined the localization of another type of receptor, p75 NTR (a similar to that for KIF16B936-GFP, localized to the tips of axons low-affinity nerve growth factor receptor) (Woo et al., 2005), in (Fig. 6C–E). Neurons expressing KIF16BFull and GFP were used control and KIF16B knockdown neurons. Immunocytochemis- as controls for somatodendritic endosomal and diffuse patterns, try showed that the number of p75 NTR clusters colocalized with respectively (statistics are presented; images of controls are not EEs was increased, whereas the density of p75 NTR clusters not displayed in Fig. 6 as they are shown in Figs. 1, 2, and 3). These colocalized with EEs was decreased, in KIF16B knockdown neu- results suggest that the second and third coiled-coil domains of rons (Fig. 5A–E,G–I). These p75 NTR clusters colocalized with the KIF16B are important for the localization of KIF16B; in their EEs and appeared to aggregate in the dendrites and the perinu- absence, KIF16B accumulates in the tips of axons. clear region of the cell body (Fig. 5C–E,G–I). This defect in To clarify whether the mislocalization of KIF16B in axonal p75 NTR localization induced by KIF16B RNAi was rescued by tips would be affected by binding to EEs, we generated a construct coexpression of RNAi-resistant KIF16B (Fig. 5F–I). that lacked the second and third coiled-coil domains but retained Next, we compared the surface expression of p75 NTR in con- the PX domain (KIF16B810-PX-GFP; Fig. 6A). This protein also trol and KIF16B knockdown neurons. Immunofluorescence moved along axonal shafts and accumulated in axonal tips (Fig. staining of cell surface p75 NTR revealed that the cell surface den- 6F,H). Using a constitutively active KIF5C motor domain and sity of the receptor was significantly reduced in KIF16B knock- the PX domain of KIF16B, we generated a chimera to determine down neurons (Fig. 5G,K). Surface biotinylation also showed whether the motor domain had any effect on the localization of that cell surface expression of p75 NTR was significantly reduced in KIF16B. Interestingly, we observed that the chimeric protein also KIF16B knockdown neurons compared with that in controls moved along axonal shafts and accumulated in their tips (Fig. (Fig. 5L,M). This reduction in surface density was largely pre- 6G,H). Analysis of the axon per dendrite signal intensity as well as vented when the KIF16B RNAi-resistant construct was cotrans- the axonal density of mutant proteins per axonal density of GFP fected with the miRNA vector (Fig. 5J–M). Together, these control revealed significant differences between controls findings suggest that the loss of KIF16B results in altered receptor (KIF16BFull and GFP), KIF16B810-PX, and chimera mutants trafficking in neurons. (Fig. 6I,J). These results demonstrate that the second and third coiled-coil domains of KIF16B are crucial for the somatoden- The second and third coiled-coils of the stalk domain are dritic localization of KIF16B. critical for the proper localization of KIF16B and early Previous studies have shown that cargo localization is regu- endosomes lated by the association and dissociation with motor proteins To identify the KIF16B domain responsible for the localization of (Glater et al., 2006; Guillaud et al., 2008; Niwa et al., 2008). If the EEs in somatodendritic areas, we generated various mutant con- association and dissociation between KIF16B and EEs determine the somatodendritic localization of EEs, then KIF16B810-PX, which mislocalizes to axons, should not alter the somatodendritic 4 localization of EEs. To test this hypothesis, we investigated the Figure 5. Loss of KIF16B alters the trafficking of p75 NTR (NGFR). A–I, miRNA and control localization of EEs in neurons expressing KIF16B810-PX. vectors were transfected into neurons, which were permeabilized and double-stained with Whereas in controls KIF16BFull and EEs were specifically local- anti-p75 NTR (low-affinitynervegrowthfactorreceptor)(red)andanti-MAP2(blue)antibodies. ized to somatodendritic regions (Fig. 7A,B), KIF16B810-PX LocalizationofEEsrevealedbytheEEmarker,GFP-2xFYVE(green).A,B,Representativeneuron drove endosomes to the tips of axons (Fig. 7A,C,D). Contrary to transfected with control miRNA shows fewer colocalized p75 NTR-EEs along the dendrite. C–E, our hypothesis, the EEs were missorted to the axonal tips in neu- Representative neuron transfected with KIF16B miRNA vector (KD). D, E, Accumulation and NTR rons expressing KIF16B810-PX (Fig. 7C,D). The mislocalized en- increased colocalization of p75 with EE clusters: along a dendrite (D) and in the perinuclear dosomes colocalized with KIF16B810-PX (Fig. 7C–E), suggesting region of the cell body of a KD neuron (E). F, Image of a representative dendrite cotransfected that the recombinant motor protein directly transported EEs into with the KIF16B miRNA ϩ KIF16B-RNAi-resistant (rescue) constructs shows rescue in the den- sity of colocalized p75 NTR-EEs along the dendrite. G–I, Quantifications of the total number of the axons. Together, these data suggest that the second and third p75 NTR clusters colocalized and not colocalized with EEs: per 50 ␮m of dendrites (G, H) and per coiled-coils of the KIF16B stalk domain are pivotal in determin- 30 ␮m 2 of soma (I). J, K, Neurons expressing the RNAi vectors were stained with anti-p75 NTR ing not only the localization of KIF16B itself, but also the soma- (red) antibody without permeabilization to visualize the cell surface clusters. J, Representative todendritic localization of EEs in neurons. dendrites expressing the control, RNAi vector, and rescue show a reduced number of surface p75 NTR clusters in KD and a recovery in rescued neurons. White dotted outlines indicate the The second and third coiled-coil domains of KIF16B work as dendriticmembranes.K,Statisticalanalysisofthenumberofsurfacep75 NTR clustersper50␮m an inhibitory domain ␮ of dendrites. Quantifications were performed with ImageJ software. Scale bars, 10 m. L, M, The preceding results suggest that the second and third coiled- Cell surface protein biotinylation analysis. L, Cell surface evaluation of proteins in KIF16B-RNAi, coils of the KIF16B stalk domain affect the localization of KIF16B. control, and rescue hippocampal neurons. M, Quantification of protein relative density using Because the localization of KIF16B810 is similar to that of con- densitometry normalized to the control. The absence of intracellular ␤-tubulin verified the purityofthecellsurfaceproteinfraction.Anti-KIF16Bwasusedasknockdownefficiencycontrol. stitutively active KIF5, we hypothesized that KIF16B810 might be Protein densities were quantified using ImageJ software. For all quantifications, error bars overactive (Jacobson et al., 2006). To clarify how the second and indicate mean Ϯ SD. Fifteen neurons for each vector from three independent transfections third coiled-coils control the localization of KIF16B and EEs, we were analyzed; Student’s t test. M, Data are from three independent experiments. **p Ͻ 0.01 conducted biochemical assays. First, we conducted a microtubule (Welch’s t test). ***p Ͻ 0.001 (Welch’s t test). cosedimentation assay to examine the binding of the mutant 5078 • J. Neurosci., March 25, 2015 • 35(12):5067–5086 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism J. Neurosci., March 25, 2015 • 35(12):5067–5086 • 5079

KIF16B proteins to MTs in COS-7 cells. Free MTs, polymerized in the dendrites, along with a relatively strong dotty signal in from brain tubulin, were added to the lysed cells. Then, the MTs axons, especially at the axonal tips (Fig. 9B–D). Together, these in were sedimented by ultracentrifugation. Initially, KIF16B810 and vivo results show that full-length KIF16B binds to dendritic mi- KIF16B1096 were tested because the former lacks the second and crotubules, and that, lacking the second and third coiled-coils, third coiled-coils and accumulates in axonal tips, whereas the KIF16B810-PX-GFP is missorted to the axonal tips. latter contains the second and third coiled-coils but does not accumulate in any region and was diffusely distributed through- Binding between the motor domain and the inhibitory out the neuron (Fig. 8A,B). Approximately half of the domain KIF16B810 sedimented with the MTs, and the remaining protein Previous studies have shown that the activity of some KIFs is was cytosolic (Fig. 8C–E). However, KIF16B1096, which pos- controlled by an auto-inhibitory mechanism, in which the tail sesses the second and third coiled-coils, did not cosediment with domain associates directly with its motor domain and suppresses the MTs and was maintained in the cytoplasmic fraction (Fig. motility when cargoes are not bound to the tail domain (Nakata 8C). Importantly, in the presence of AMP-PNP, which is a and Hirokawa, 2003; Verhey and Hammond, 2009; Nakata et al., nonhydrolyzable analog of ATP and a competitive inhibitor of 2011). Thus, to gain insight into the molecular mechanisms un- ATP-dependent enzyme systems, a considerable amount of derlying the inhibition mediated by the second and third coiled- KIF16B1096 cosedimented with MTs (Fig. 8D,E). The construct coil domains, we used a coimmunoprecipitation assay to test that lacked the second and third coiled-coils but contained the whether these coiled-coils (ID) of the stalk region of KIF16B PX domain (KIF16B810-PX) showed almost the same level of could bind directly to the motor domain to inhibit its activity. MT binding as KIF16B810. Furthermore, neither KIF16B810 nor Using this assay, we found that the ID binds directly to the motor KIF16B810-PX showed a significant difference in binding to MTs domain of KIF16B (Fig. 10A,C). We also examined whether the in the presence or absence of AMP-PNP (Fig. 8C–E). Without motor domain interacts with the PX domain. We observed no additives, full-length KIF16B bound to MTs less than KIF16B810 binding between the motor and PX domains of KIF16B, as ex- or KIF16B810-PX, whereas in the presence of AMP-PNP, all pected based on previous results (Fig. 10B,C). To analyze the role three proteins exhibited a similar binding ability. Our negative of the ID within the cell, we overexpressed the ID in COS-7 cells control, Myosin Vb, did not bind to MTs (Fig. 8C–E). Therefore, and hippocampal neurons. In COS-7 cells that were transfected the cosedimentation assay results suggest that the second and with the GFP control, EEA1 staining was evenly distributed third coiled-coil domains inhibit binding of KIF16B to microtu- throughout the cytoplasm and in the somatodendritic regions of bules in the presence of ATP. neurons, with an even distribution of puncta. When the ID was Next, we examined the microtubule binding ability of KIF16B expressed, the endosomes accumulated in the center of COS-7 in living neurons. Cultured hippocampal neurons were trans- cells; and in hippocampal neurons, the EEs tended to aggregate in fected with KIF16BFull-GFP or KIF16B810-PX-GFP. To visual- the somatodendritic regions (Fig. 10D–G). We further conducted ize the microtubule-bound population, transfected cells were live imaging to test whether the ID affected the motility of KIF16B transiently permeabilized with the bacterial toxin streptolysin-O, or EEs in neurons. Cotransfection of GFP-2xFYVE with the ID in which is a membrane-damaging protein toxin that creates tran- neurons disturbed the motility of EEs in the somatodendritic sient pores in cell membranes by binding to the cholesterol- regions compared with that in controls (the percentage of motile containing target membranes. Washing removed those proteins EEs: control, 81%, ϩ ID, 29%; Fig. 10H–L). These cell culture not bound to MTs from the cytoplasm. After 10 min, the neurons results, together with the coimmunoprecipitation data, indicate were monitored using a special BiG (GaAsP) detector attached to that the ID binds to the motor domain and suppresses the motil- a LSM710 Duo laser scanning confocal microscope. KIF16BFull- ity of KIF16B and the EEs. GFP displayed a filamentous distribution pattern in the den- The microtubule cosedimentation assay findings suggested drites, suggesting that it was bound to dendritic MTs (Fig. 9A). In that the inhibition of the binding of KIF16B to microtubules is contrast, KIF16B810-PX-GFP showed a weak and diffuse signal regulated by ATP (Fig. 8C–E). Consequently, it is possible that the binding between the ID and the motor domain may also 4 depend on ATP. To test this hypothesis, we examined the binding of a GST-tagged ID to a GFP-tagged motor domain, the PX do- Figure 6. Second and third coiled-coils of the KIF16B stalk domain are critical for the soma- main, or full-length KIF16B using affinity chromatography. The todendritic localization of KIF16B. A, Schematic representation of the deletion and chimera motor domain and full-length KIF16B were able to bind to the ID mutants. B–J, Hippocampal neurons were transfected with the indicated constructs, cultured in the presence of ATP but not in the presence of AMP-PNP, for 10–12 h, fixed, and then stained with a dendrite marker (MAP2) and a neuronal marker (tubulin-␤3). B, Representative image of a mutant lacking the third coiled-coil (KIF16B936) whereas the PX domain did not bind to the ID (Fig. 10M–O). that accumulated at axon tips. C, KIF16B810-GFP lacking the second and third coiled-coils Consistent with the results of the cosedimentation assay, full- localizes in the tips of axons. B, C, Enlarged merged insets and arrows indicate axonal tips as length KIF16B showed slightly weaker binding to the ID com- revealed with only tubulin-␤3 staining. D, A shorter 4 h expression of KIF16B810 mutant also pared with the motor domain in the presence of ATP. Therefore, results in diffuse expression in dendrites and specific localization at axon tips. E, Ratio of axonal our data suggest that ATP regulates not only the inhibition of tips per axonal shafts signal intensity for the KIF16B810, KIF16B936, and controls (KIF16BFull binding of KIF16B to microtubules, but also binding of the ID to and GFP). F, KIF16B810-PX-GFP is localized to axonal shafts and axonal tips. G, Chimera the motor domain of KIF16B. (KIF5C560-KIF16BPX-GFP) shows a similar localization pattern in axonal shafts and tips. F, G, High-magnification merges and arrows indicate axonal localization as revealed with only Discussion tubulin-␤3staining.DendritesarelabeledwithMAP2.Scalebars,10␮m.H–J,Quantifications Impact of KIF16B on EE and receptor trafficking in neurons of KIF16B810-PX, chimera, and controls (KIF16BFull and GFP). H, Relative axonal fluorescence intensity normalized to their background signals. I, Arbitrary units of axons per dendrites. J, We examined the role of KIF16B in the transport of EEs in AxonaldensityoftheproteinofinterestperaxonaldensityofGFPcontrol.DataaremeanϮSD. neurons. Our results revealed that KIF16B transports EEs in Twenty-one neurons from three independent transfections were examined. ***p Ͻ 0.001 (E, somatodendritic regions. When KIF16B is knocked down, the Student’sttest;ANOVAandposthoctest).n.s.,Notsignificant.Signalintensityanddensitywere bidirectional movements of EEs are perturbed, and they accumu- analyzed using ImageJ software. late in the cell body and dendrites. This phenotype was rescued by 5080 • J. Neurosci., March 25, 2015 • 35(12):5067–5086 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism

Figure7. Secondandthirdcoiled-coilsoftheKIF16Bstalkdomainpotentiatethesomatodendriticlocalizationofearlyendosomes.A–E,TheindicatedIRES-containingconstructswereexpressed in hippocampal neurons, which were cultured for 10–12 h, fixed and immunostained with anti-MAP2 antibody (blue). Localization of EEs was revealed by GFP-2xFYVE in KIF16BFull-FLAG control (B) and in KIF16B810-PX-FLAG-transfected neurons (C, D). D, Lower-magnification image showing that both GFP-2xFYVE and KIF16B810-PX-FLAG misaccumulated at axonal tips. Insets (of dotted areas),ColocalizationofEEswithKIF16BFullinsomatodendriticregionsofBandKIF16B810-PXalongtheaxoninCanddistalaxoninD.Whitearrowsindicatetheaxonalshafts.Bluearrowsindicate axonaltips.Scalebars,10␮m.E,PercentageoftheaxonalcolocalizationofKIF16BFull(control)andKIF16B810-PXwithEEswasquantifiedwithapixel-by-pixelcorrelationbetweengreenandred signals using ImageJ software. This revealed a significant mislocalization of the EEs with KIF16B810-PX along the axons and tips. Error bars indicate mean Ϯ SD. Twenty-one neurons from three independent preparations were examined. ***p Ͻ 0.001 (Student’s t test).

KIF16B expression. Our results suggest that the normal activity of tern, suggesting that the PX domain of KIF16B is the cargo- KIF16B keeps the EEs moving properly and dispersed along the binding domain. dendrites. The bidirectional motion of EEs and KIF16B is consis- Previous studies have proposed that EEs can acquire and re- tent with the bidirectional arrangement of MTs in somatoden- cycle cargo to the cell membrane as long as KIF16B keeps them dritic regions. Considering that KIF16B knockdown has the same near the surface of HeLa cells (Lin et al., 2002; Hoepfner et al., effect on the motility of EEs in both anterograde and retrograde 2005). EEs also contribute to the somatodendritic and axonal directions, we suggest that, relative to MTs, velocity distribution localization of receptors by participating in somatodendritic- is independent of direction and may depend on cargoes carried in specific endocytosis, recycling, and transcytosis processes that EEs and/or on associated proteins (Gasman et al., 2003). We lead to the redistribution of receptors from dendrites to axons found that disruption of the PX domain abrogates the binding of (Yap et al., 2008; Winckler and Mellman, 2010; Petersen et al., KIF16B to EE. However, expression of the PX domain on its own 2014). Here, we found that defects in KIF16B expression, and the results in a phenotype resembling the wild-type endosomal pat- corresponding EE motility, perturb the distribution of GluR1 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism J. Neurosci., March 25, 2015 • 35(12):5067–5086 • 5081

Figure8. Secondandthirdcoiled-coilsofKIF16Bfunctionasaninhibitorydomain.A,B,LocalizationofKIF16BmutantsinhippocampalneuronsstainedwithMAP2andtubulin-␤3.KIF16B810- GFPislocalizedattipsofaxons,whereasKIF16B1096-GFPshowsdiffuselocalization.Arrowsindicatetipsofaxons.MAP2andtubulin-␤3imagesarenotshownherebecauseoftheinterferencewith the grayscale images. Scale bars, 10 ␮m. Representative images from 21 neurons obtained from three independent preparations are shown. C–E, Microtubule cosedimentation assay. C, D, COS-7 cells were transfected with KIF16B810, KIF16B1096, KIF16B810-PX, KIF16BFull, or myosin Vb, tagged with GFP, lysed, mixed with tubulin, incubated at 37°C, and centrifuged to separate cytosolic and microtubule pellet fractions. C, In the absence of AMP-PNP. D, In the presence of AMP-PNP. Western blot analysis was performed with anti-GFP and anti-␣-tubulin antibodies. Myosin Vb was used as a negative control. E, Quantification of relative band intensities corresponding to the Western blots in C, D was performed using ImageJ software. Error bars indicate SD. Four individual experiments were conducted for each value. *p Ͻ 0.05 (ANOVA and post hoc test). **p Ͻ 0. 01 (ANOVA and post hoc test). ***p Ͻ 0.001 (ANOVA and post hoc test). n.s., Not significant. 5082 • J. Neurosci., March 25, 2015 • 35(12):5067–5086 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism

Figure 9. KIF16B binds to dendritic microtubules. A–D, Live neuron microtubule binding assay. Neurons, expressing KIF16B GFP-tagged constructs for 10–12 h, were transiently permeabilized with streptolysin-O and monitored after 10 min using a laser scanning confocal microscope equipped with a special detector. A, Neuron expressing KIF16BFull shows microtubule pattern along the dendrite. Right, 3D profile of the fluorescence intensity along the dendrite showing dotty signals. B, C, Neuron expressing KIF16B810-PX. B, Weak and diffuse signals along the dendrite. Right, 3D profileofthefluorescenceintensityalongthedendritealsoindicatesweakanddiffusesignal.C,Strongsignalsinaxonaltips.Righttop,3Dprofileofthefluorescenceintensityinthedistalaxonsand axonal tips presenting dotty signals. Right bottom, Magnification of the distal portion of the axon. Scale bars, 10 ␮m. 3D fluorescence profiles were obtained using Carl Zeiss LSM image software. D,StatisticalanalysisofsignalratioofdistalaxonsperaxonshaftsforKIF16B810-PXandKIF16BFull.ImageswerequantifiedwithImageJsoftware.DataaremeanϮSD.Fifteenneuronsfromthree independent transfections were examined. ***p Ͻ 0.001 (Student’s t test). and p75 NTR in neurons. The electrophysiological function of Previous studies elucidated several mechanisms that deter- AMPA receptors, critical for neuronal plasticity, was consistently mine the localization of molecular motors and showed that the impaired (Whitlock et al., 2006; Shepherd and Huganir, 2007; motor domain determines the distribution of KIFs (Nakata Kessels and Malinow, 2009). Moreover, surface expression of and Hirokawa, 2007; Nakata et al., 2011; Huang and Banker, these receptors was reduced, resulting in decreased amplitude 2012). Some motors specifically travel in axons, whereas oth- and increased interevent interval of mEPSCs in KIF16B knock- ers move in axons and dendrites. It has been suggested that down neurons. These results revealed that KIF16B is important cargo-binding affects the localization of some motors (Setou for surface localization of GluR1 and p75 NTR. KIF16B may be et al., 2002; Hoogenraad et al., 2005; Jenkins et al., 2012). We involved in regulating trafficking of many functionally important show here, for the first time, that the stalk domain of a kinesin receptors through its activity in transporting EEs in somatoden- has the capacity to potentiate the particular localization of that dritic areas. Collectively, our findings indicate a functional sig- motor protein and its cargo. nificance of KIF16B in EE transport and receptor trafficking in The cargo-binding domains of some KIFs bind directly to the cell bodies and dendrites of neurons. motor domain and inhibit motor activity when unbound to cargo (Verhey and Hammond, 2009). This mechanism is called New regulatory mechanism potentiates the selective “tail-inhibition.” In addition to this mechanism, we suggest a distribution of KIF16B and EEs new “stalk inhibition” mechanism. Deleting the second and third Our findings show that KIF16B and its cargo of EEs are specifi- coiled-coils of the KIF16B stalk domain mislocalized KIF16B cally localized in somatodendritic regions of neurons. KIFs are (Fig. 6). Furthermore, although the wild-type KIF16B was pre- key regulators that determine the destination of cargo proteins. cisely localized in somatodendritic regions, KIF16B missing the Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism J. Neurosci., March 25, 2015 • 35(12):5067–5086 • 5083 5084 • J. Neurosci., March 25, 2015 • 35(12):5067–5086 Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism second and third coiled-coils and its cargo of EEs accumulated dissociation of KIF16B with EEs, but rather by an intramolec- specifically in the tips of axons (Figs. 7 and 9). Biochemical and ular regulatory mechanism. cell biological assays revealed that the second and third coiled- Disruption of the stalk inhibition mechanism mislocalizes coils bound directly to the motor domain and inhibited the at- KIF16B into axons, and KIF16B motor-containing constructs tachment of the motor with MTs in an ATP-dependent manner without complete ID and PX domains also show axonal mislo- (Figs. 8 and 10). These results suggest that the motor activity of calization. This suggests that the motor domain itself has a ten- KIF16B is negatively regulated by the second and third coiled-coil dency to localize in axons. In wild-type neurons, the presence of domains (i.e., the ID). The requirement of ATP for stalk inhibi- the ID and stalk inhibition mechanism prevent the motor from tion in KIF16B is similar to that of the kinesin-5 tail-inhibition being distributed in areas of the neuron not requiring the motor mechanism. The IAK region of KIF5 inhibits the ATPase activity (i.e., the axon) and, as a result, potentiate proper somatoden- of the kinesin, resulting in a weak affinity for MTs in the presence dritic localization of KIF16B and EEs. Our data suggest that stalk of ATP. This arises from the selective inhibition of the release of inhibition is turned off in the cell body and dendrites, which ADP upon the initial interaction with an MT (the ADP state of leads one to question why stalk inhibition does not function in the head is “turned off”) (Hackney and Stock, 2000). Thus, the somatodendritic regions. Because wild-type KIF16B-carrying two coiled-coil sections of the stalk are essential for the KIF16B EEs do not enter the axon but travel in somatodendritic areas, autoregulatory mechanism that inhibits microtubule binding in it is unlikely that the binding of EE to the PX domain of the presence of ATP. KIF16B represses the ID/motor domain interaction and turns The mechanisms by which organelles are sorted are un- on the motor. Given our data for the diffuse distribution of clear. Some cargoes are directly sorted to axons after modifi- KIF16B1096 and KIF16BPXmut throughout the neuron as cation in the Golgi apparatus (Nakata and Hirokawa, 2003), well as our data showing that the ID disturbs the motility of the and a diffusion barrier at the axon initial segment keeps den- motor in somatodendritic regions, it is likely that the binding dritic proteins from mislocalizing to axons (Song et al., 2009). of the ID and motor domain occurs throughout the neuron, In addition, the molecular mechanisms determining the so- making it improbable that an axon-specific factor exists to matodendritic localization of EEs remain unknown. Previous enhance the ID/motor interaction and switch off the motor in studies have shown that cargo transport is regulated by the axons. Thus, because functional KIF16B moves along MTs and association and dissociation of motors (Bridgman, 1999; Guil- transports EEs in somatodendritic regions and because the laud et al., 2008; Macaskill et al., 2009, Wang and Schwarz, presence of the ID is necessary for this selective localization, 2009; Jenkins et al., 2012). However, our results suggest that we propose the presence of a somatodendritic-specific factor the association of the PX domain of KIF16B with EEs is not (SD-ID factor) that binds to the ID, turns off stalk inhibition, inhibited in axons because the expression of KIF16B810-PX- and triggers KIF16B movement along MTs in somatodendritic GFP (KIF16B⌬ID), which possesses the ability to associate areas (Fig. 10P). Considering the pull-down assay result, we with EEs, missorts both to axonal tips. This suggests that the conjecture that the association of an SD-ID factor with the ID localization of EEs is not controlled by the association and is regulated by an ATP-dependent system. Kinesin-associated 4 proteins are known to regulate the activity of a variety of KIFs. Consequently, this SD-ID factor is likely an ID-associated pro- Figure10. MotordomainofKIF16Bbindstotheinhibitorydomain.A–C,Coimmunoprecipi- tein located in somatodendritic regions. Further biochemical tations of the motor domain with the second and third coiled-coils (ID) of the KIF16B stalk studies are needed to determine the identity of this factor. region(A)andthePXdomainofKIF16B(B).Proteinsweretransfectedandprecipitatedwithan Considering that KIF16B1096 and KIF16BPXmut, which anti-GFP antibody. Western blot analysis of input and pellet fractions was performed using were unable to bind to EEs, were diffusely localized throughout anti-GFPandanti-FLAGantibodies.Arepresentativeblotisshown.C,QuantificationofWestern the neuron, the binding of EE to KIF16B may spatially strengthen blotting band signals for the coimmunoprecipitations. D–L, The inhibitory domain disrupts the movements of KIF16B and the EEs. D–F, EGFP or EGFP-ID were transfected into COS-7 cells (D) the SD-ID/ID interaction. The SD-ID/ID interaction suppresses and hippocampal neurons (E), which were stained with anti-EEA1 and, for neurons, also with stalk inhibition, enabling KIF16B to move along MTs in somato- MAP2. F, Percentage of EEs distribution per COS-7 cells. G, Relative signal density of EEs in 500 dendritic regions. Additionally, full-length KIF16B displayed a pixels of hippocampal neurons (hip.). Quantifications revealed the accumulation of EEs in cells weak affinity for MTs in the cosedimentation assay and weaker expressing the ID compared with control. Distribution and density measured with ImageJ soft- binding to the ID in the pull-down assay. This suggests that, as ware. H–L, Analysis of EE movements in the presence or absence of the ID. Time-lapse images no direct attachment was detected between the PX and motor (H) and kymograph (I) of the control neuron that expressed the EE marker, FYVE, and shows domains or ID, the presence of the PX domain may reduce the bidirectional movement of EEs along the dendrite. Arrows indicate an example of a moving EE. interaction of the motor domain with the ID, thereby bringing Time-lapse images (J) and kymograph (K) of the neuron that was cotransfected with ID and the ID into contact with the SD-ID factor and allowing move- FYVE displays significantly reduced bidirectional movement of the EEs along the dendrite. Ar- rows point to an example of a stalled EE. Scale bars, 10 ␮m. L, Classification of motility. p Ͻ ment along MTs. Therefore, in wild-type KIF16B, the presence 0.001.M–O,GSTaffinitychromatographyanalysisforinteractionsbetweenGST-IDwithKIF16B of the PX domain and the binding of EE to the PX domain fragments tagged with GFP. M, Coomassie blue-stained gels indicating the level of GST and presumably enhance the association of SD-ID factor with GST-inhibitory domain on the beads. N, GST pull-down assay between GST and GST-ID with the ID. KIF16B810-GFP (motor domain), full-length KIF16B, and KIF16BPX as a control in the presence This study presented a novel “stalk inhibition” mechanism of ATP or AMP-PNP. O, Statistical analysis of Western blotting results in the GST pull-down that regulates the potential of KIF16B to determine the selective assay.Forallstatisticalanalyses,errorbarsindicatemeanϮSDfromthreeindependentexper- localization of this motor protein and its EE cargo in somatoden- ϭ iments. A total of 18 COS-7 cells (F) and 18 neurons (G, L) were analyzed. C, O, n 3. n.s., Not dritic regions of neurons. The interaction between the stalk in- significant.*pϽ0.05(Student’sttest).**pϽ0.01(Student’sttest).***pϽ0.001(Student’s t test). L,␹ 2 test. P, Diagram model summarizing the “stalk inhibition” regulatory mechanism. hibitory and motor domains may serve as a switch to control the KIF16BbindingtoMTsissuppressedbytheinteractionofthesecondandthirdcoiled-coilsofthe binding of the KIF16B motor to microtubules. Our proposed stalk domain (ID) and motor domain in the presence of ATP. KIF16B moves on MTs and trans- SD-ID factor suppresses stalk inhibition by associating with the ports early endosomes in somatodendritic regions as long as the SD-ID factor is associated with ID in somatodendritic regions, resulting in movement along the ID in an ATP-dependent system and represses motor and ID interaction. microtubules. Farkhondeh et al. • KIF16B in Neurons and “Stalk Inhibition” Mechanism J. Neurosci., March 25, 2015 • 35(12):5067–5086 • 5085

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